Gene structure and expression analysis of the drought- and abscisic acid-responsive CDeT11-24 gene family from the resurrection plant Craterostigma plantagineum Hochst

In order to understand the molecular mechanisms which are responsible for desiccation tolerance in the resurrection plant Craterostigma plantagineum Hochst. a thorough analysis of the CDeT11-24 gene family was performed. CDeT11-24 comprises a small gene family whose genes are expressed in response to dehydration, salt stress and abscisic acid (ABA) treatment in leaves. The gene products are constitutively expressed in roots and disappear only when the plants are transferred to water. It is therefore suggested that the proteins are involved in sensing water status. The predicted proteins are very hydrophilic; they share some features with late-embryogenesis-abundant proteins, and sequence similarities were found with two ABA- and drought-regulated Arabidopsis genes. The analysis of β-glucuronidase reporter genes driven by the CDeT11-24 promoter showed high activity in mature seeds in both transgenic Arabidopsis and tobacco. In vegetative tissues the promoter activity in response to ABA was restricted to young Arabidosis seedlings. The responsiveness to ABA during later developmental stages was regained in the presence of the Arabidopsis gene product ABI3. Dehydration-induced promoter activity was only observed in Arabidopsis leaves at a particular developmental stage. This analysis indicates that some components in the signal transduction pathway of the resurrection plant are not active in tobacco or Arabidopsis. Received: 26 April 1997 / Accepted: 16 July 1997     

Expression of desiccation-related proteins from the resurrection plant Craterostigma plantagineum in transgenic tobacco

Three cDNAs encoding desiccation-induced proteins from the resurrection plant Craterostigma plantagineum were each ligated to a triplicated CaMV 35S promoter and a nopaline synthase 3-flanking region in an Agrobacterium vector and introduced into tobacco. Transgenic plants expressed the encoded Craterostigma proteins at high levels. This did not lead to changes in the phenotype, in the growth habit or in basic photosynthetic parameters. In tobacco, one protein was targeted to the chloroplast stroma which is its normal location in Craterostigma. These desiccation-related proteins are not sufficient per se to increase drought tolerance as measured by ion-leakage tests.     

Desiccation of the resurrection plant Craterostigma plantagineum induces dynamic changes in protein phosphorylation

Reversible phosphorylation of proteins is an important mechanism by which organisms regulate their reactions to external stimuli. To investigate the involvement of phosphorylation during acquisition of desiccation tolerance, we have analysed dehydration-induced protein phosphorylation in the desiccation tolerant resurrection plant Craterostigma plantagineum. Several dehydration-induced proteins were shown to be transiently phosphorylated during a dehydration and rehydration (RH) cycle. Two abundantly expressed phosphoproteins are the dehydration- and abscisic acid (ABA)-responsive protein CDeT11-24 and the group 2 late embryogenesis abundant (LEA) protein CDeT6-19. Although both proteins accumulate in leaves and roots with similar kinetics in response to dehydration, their phosphorylation patterns differ. Several phosphorylation sites were identified on the CDeT11-24 protein using liquid chromatography-tandem mass spectrometry (LCMS/MS). The coincidence of phosphorylation sites with predicted coiled-coil regions leads to the hypothesis that CDeT11-24 phosphorylations influence the stability of coiled-coil interactions with itself and possibly other proteins.     

An effective and reproducible transformation protocol for the model resurrection plant Craterostigma plantagineum Hochst.

Procedures previously established for plant regeneration and Agrobacterium tumefaciens-mediated genetic transformation of the desiccation-tolerant plant, Craterostigma plantagineum, have been further developed. A highly effective tissue culture system was established based on the integrated optimisation of antioxidant and growth regulator composition and the stabilisation of the pH of the culture media by means of a potassium phosphate buffer. The undesirable hyperhydricity of Craterostigma tissue in tissue culture was also circumvented by these modifications, which serve as an alternative to the previously described procedures. The high efficiency of plant regeneration from the callus phase provided the basis for optimising genetic transformation in Craterostigma. For gene delivery, both a standard (method A) and a modified protocol (method B), the latter having previously resulted in successful Agrobacterium-mediated transformation of monocot cereals, were applied. Physical and biochemical key variables in transformation were evaluated, such as gene gun-mediated microwounding of plant explants, infiltration of Agrobacterium suspension cultures into target tissues and the influence of in vitro pre-induction of vir genes. While the physical enhancement of Agrobacterium infection (microwounding, infiltration) had no positive effect, the in vitro pre-induction of vir genes (biochemical enhancement) resulted in a twofold increase in the transformation frequency as compared to the conventional protocol (method A).     

Constitutive expression of small heat shock proteins in vegetative tissues of the resurrection plant Craterostigma plantagineum

Using antibodies raised against two sunflower small heat shock proteins (sHSPs), we have detected immunologically related proteins in unstressed vegetative tissues from the resurrection plant Craterostigma plantagineum. In whole plants, further accumulation of these polypeptides was induced by heat-shock or water-stress. In desiccation-intolerant Craterostigma callus tissue, we failed to detect sHSP-related polypeptides, but their expression, and the concurrent acquisition of desiccation tolerance was induced by exogenous abscisic acid (ABA) treatment. In untressed plants, the cross-reacting polypeptides were abundant in the roots and lower part of the shoots, where they showed homogeneous tissue-distributions. This constitutive expression is novel for vegetative tissues of higher plants, and resembles the expression patterns of sHSPs in desiccation-tolerant zygotic embryos and germinating seeds.J.A. and C.A. contributed equally to this work and are both considered to be first author